Pulmonary arterial hypertension (PAH) is an occlusive disease of the pulmonary arteries leading to serious hemodynamic abnormality, right heart failure, and premature death. The molecular mechanisms behind PAH are still unclear. Without a more complete understanding of PAH and how its complex vascular dysfunctions relate to one another, patients will suffer from imprecise diagnosis and drug therapy that may be less than optimal. Despite recent advances and introduction of new clinically approved drugs, the 5-year survival from pulmonary hypertension remains an estimated 50% (Archer and Rich, 2000). Consequently, treatment for PAH, while recently improved, still offers significant and long-lasting improvement in only a minority of patients. A methodology to elucidate the molecular pathways associated with PAH could guide the development of new therapies for this disease.
Though platelets and other cells may have a role in PAH, pulmonary endothelial cells and pulmonary smooth muscle cells appear to be the primary sites of disease progression (Humbert et al 2004). Molecular pathways that show abnormality in pulmonary endothelial cells and pulmonary smooth muscle cells during PAH include endothelin-1 (Giaid et al 1993), serotonin & serotonin transporter (Marcos et al 2003), thromboxane (Walmrath et al 1997), nitric oxide synthase (Kobs and Chesler 2006), prostacyclins (Gailes, et al 2001), potassium channels (Mandegar et al 2002), BMP signaling (Eddahibi et al 2002), and survivin (McMurtry et al 2005). PAH impairs normal signaling and growth in both pulmonary endothelial and pulmonary smooth muscle cells, yet the cellular abnormalities seem to shift over time in unpredictable patterns that has thus far escaped concise definition (Michelakis, 2006).
PAH may be understood as proceeding in phases. In early PAH, endothelial apoptosis occurs, probably resulting in pulmonary arteriole plugging and an increase in pulmonary vascular pressure (Michelakis, 2006). In late PAH, chronic exposure to elevated pulmonary artery pressure together with dysfunctional endothelial signaling initiates hyperproliferation of smooth muscle cells (McMurtry et al 2005). Increased concentric pulmonary smooth muscle cell proliferation leads to ever increasing pulmonary artery pressure, right ventricular failure, and death.
Lung pathology in all PAH patients show thickening throughout the arterial wall of the pulmonary vascular bed. In some forms of the disease, the pulmonary vascular lesions are reversible (e.g. in newborns with congenital heart defects). In other patients, such as those with the idiopathic form, the lesions are irreversible. It is unknown how these variations in PAH relate to one another on a molecular basis (Pearl et al 2002).
Current therapies for PAH patients primarily target vascular tone. Treatments that aim at correcting potassium channel dysfunction (Machado et al 2001), nitric oxide impairment (Humbert et al 2004), prostacyclin impairment (Tuder et al 1999, Christman et al 1992), and endothelin-1 expression (Giaid et al 1993) have all been clinically available for several years. These therapies offer some relief from hemodynamic symptoms, but most patients show only a transient response. The proliferative disease continues to progress in most PAH patients, resulting in a five year mortality rate that remains at around 50% (Newman et al 2004).
Currently, there are no clinically available routine means to obtain endothelial and smooth muscle samples from the pulmonary arteries of pulmonary hypertension patients for diagnosis, disease staging or drug discovery. Applicant's earlier invention, described in U.S. Pat. No. 5,406,959, describes an endoarterial biopsy catheter that has demonstrated its safety and effectiveness in normal canines (Rothman, Mann et al., 1996), canines with experimentally-induced pulmonary hypertension (Rothman, Mann et al., 1998), and canines with single-sided lung transplant rejection (Rothman, Mann et al., 2003). Preliminary studies have also demonstrated the safety and efficacy of a catheter-based method to obtain endovascular samples from a porcine model of PAH.
Percutaneously-obtained pulmonary endoarterial biopsy samples were found to be of sufficient quantity and quality for porcine whole genome mRNA microarray analysis and microRNA analysis. Whole genome microarray analysis revealed time-sensitive variations in gene expression values as PAH progressed in the subject animal model. Genes previously shown to be associated with PAH displayed changes characteristic of the disease, and genes previously unassociated with PAH also displayed expression level dysregulation. These findings raise the possibility that the endoarterial biopsy catheter combined with microarray analysis may provide a valuable platform for the discovery of novel drug and biomarker targets in pulmonary hypertension and a platform to deliver individualized pharmacotranscriptomics.
MicroRNA analysis revealed pressure sensitive changes in microRNA expression. As our surgical shunt model of pulmonary hypertension progressed from a high flow low pressure (HFLP) manifestation to a high flow high pressure (HFHP) manifestation, different microRNAs became dysregulated either increasing or decreasing in expression relative to our baseline normal values.
Most new therapies promise to focus on arresting either the endothelial apoptosis that characterizes early PAH (angiopoetin-1 & endothelial nitric oxide synthase cell-base gene transfer (Zhao et al 2003; 2005), caspase inhibitors (Taraseviciene-Stewart at al 2001)) or the smooth muscle cell proliferation typical of late PAH (dichloroacetate (McMurtry et al 2004), simvastatin (Nishimura et al 2003), sidenafil (Wharton et al 2005), imatinib (Schermuly et al 2005), anti-survivin (McMurtry et al 2005), K+ channel replacement gene therapy (Pozeg et al 2003)).
Before administering therapies, however, it would be extremely valuable to determine which genes are dysregulated in each PAH patient at any stage of their individual disease progression. Without knowing what genes are aberrant during any point in the patient's disease course, targeted therapies may miss the mark in some patients. Life threatening side effects may emerge if the wrong cells, at the wrong time, are encouraged to die or proliferate in patients with compromised pulmonary vascular health.
A powerful method for determining the gene expression levels of thousands of genes simultaneously are DNA microarrays. Initially used for the classification of cancers that were difficult to discriminate histologically (Golub et al 1999, Bhattacharjee et al 2001, and Ramaswamy et al 2001), microarrays have been more recently applied to PAH (Geraci et al 2001). PAH microarray studies have been performed on whole lung homogenates in humans (Fantozzi et al 2005) and rats (Hoshikawa et al 2003), surgically-dissected pulmonary arteries in pigs (Medhora et al 2002), laser-microdissected pulmonary arteries in rats (Kwapiszewska et al 2005), and mononuclear peripheral blood in humans (Bull et al 2004). These studies have been performed to discover potentially novel PAH disease pathways, biomarkers, therapeutic targets and patient classification gene expression profiles.
To advance PAH microarray studies into practical clinical use, tissue procurement methodologies are required that do not require surgical explant or postmortem procurement, and peripheral blood has thus far proven to be inadequate to discriminate gene expression signatures between subgroups of PAH patients (Bull et al 2004; Bull et al 2007). To take advantage of the full power of microarray technologies in PAH patients, a safe and effective minimally invasive means for the repeat procurement of endovascular samples from living PAH patients is required.
The present invention provides for the use of a novel interventional catheter, an endoarterial biopsy catheter, to obtain serial biopsy specimens from hypertensive pulmonary vessels for analysis. The ability to procure endothelial and smooth muscle samples in a minimally invasive manner will allow physicians to use microarray profiling and other techniques to classify patients upon initial presentation according to their gene expression signatures, prescribe therapies that target genes empirically found to be dysregulated in each individual patient, and monitor and adjust PAH patient therapy according to subsequent biopsy findings. A greater understanding of the complex molecular pathways underlying each patient's PAH should enable more precise diagnosis and the delivery of more effective therapies. Also of importance is the ability to discover new uses for existing drugs as well as discovering new drug targets.
Individualized pharmacotranscriptomics based on endoarterial biopsy and microarray analysis represents a reasonable choice for researchers struggling with the complexities and contradictions of PAH and other vascular diseases. The huge literature generated from in vitro and animal studies falls short, at times, in addressing the actual facts of patient health. Many commentators describe this dilemma as the “bench-to-bedside gap”, where in vitro and animal laboratory data fails to model human disease circumstances (Aird, 2004). Bridging that gap through catheter-based access to the vasculature in a model that recapitulates the clinical and histopathological manifestations of a form of human pulmonary hypertension will likely enable closer correlations between animal studies and patient care, and serve as a model for other vascular-based diseases such as atherosclerosis, congestive heart failure, sickle cell disease, organ transplant rejection, connective tissue diseases, chronic obstructive pulmonary disease, pulmonary embolism, asthma, systemic inflammatory response, battlefield trauma, cancer, sepsis and acute respiratory distress syndrome. There is a need in the art to provide data from gene expression analyses in order to target novel candidates for use in treating or preventing PAH.